Artificially engineered protein hydrogels to mimic nucleoporin selective gating

ABSTRACT

Disclosed are synthetic polypeptides modeled after NspI nucleoporin which are useful for forming hydrogels characterized by selective permeability. The polypeptides and hydrogels formed from them include phenylalanine-glycine (FG) repeats, which are believed to participate in the selectivity of the nuclear pore complex. Also disclosed are filtering devices, drug delivery devices, and methods of separating or selectively filtering macromolecules using the hydrogels.

RELATED APPLICATIONS

This application is the U.S. National Stage of PCT/US2015/036739, filedJun. 19, 2015, which claims benefit of priority to U.S. ProvisionalPatent Application No. 62/015,012, filed Jun. 20, 2014.

GOVERNMENT SUPPORT

This application is the U.S. National Stage of PCT/US2015/036739, filedJun. 19, 2015, which claims benefit of priority to U.S. ProvisionalPatent Application No. 62/015,012, filed Jun. 20, 2014.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 22, 2017, isnamed MTV-15601_SL.txt and is 69,998 bytes in size.

BACKGROUND OF THE INVENTION

The entry and exit of large molecules from the eukaryotic cell nucleusis tightly controlled by nuclear pore complexes (NPCs). Although smallmolecules can enter and exit the nucleus without regulation,macromolecules such as RNA, mRNA, ribosomal proteins, DNA polymerase,lamins, carbohydrates, signaling molecules, and lipids requireassociation with karyopherins called importins to enter the nucleus andexportins to exit.

Nuclear pore complexes are large protein complexes that span the nuclearenvelope, which is the double membrane surrounding the eukaryotic cellnucleus. The proteins that make up the nuclear pore complex are known asnucleoporins.

Nucleoporins are only required for the transport of large hydrophilicmolecules above 40 kDa, as smaller molecules pass through nuclear poresvia passive diffusion. For example, nucleoporins play an important rolein the transport of mRNA from the nucleus to the cytoplasm aftertranscription. Depending on their function, certain nucleoporins arelocalized to a single side of the nuclear pore complex, either cytosolicor nucleoplasmic. Other nucleoporins may be found on both faces.

There are three distinct types of nucleoporins, each having a uniquestructure and function. These three types are structural nucleoporins,membrane nucleoporins, and FG-nucleoporins.

Structural nucleoporins form the ring portion of the NPC. They span themembrane of the nuclear envelope and are often referred to as thescaffolding of a nuclear pore. Structural nucleoporins come together toform Y-complexes that are composed of seven nucleoporins. Each nuclearpore contains sixteen Y-complexes for a total of 112 structuralnucleoporins.

Membrane nucleoporins are localized to the curvature of a nuclear pore.These proteins are embedded within the nuclear membrane at the regionwhere the inner and outer leaflets connect.

FG-nucleoporins are so named because they contain repeats of the aminoacid residues phenylalanine (F) and glycine (G). FG repeats are smallhydrophobic segments that break up long stretches of hydrophilic aminoacids. These FG-repeat segments are found in long random-coil portionsof the protein which stretch into the channel of nuclear pores and arebelieved to be primarily responsible for the selective exclusivity ofnuclear pore complexes. These segments of FG-nucleoporins form a mass ofchains which allow smaller molecules to diffuse through but excludelarge hydrophilic macromolecules. These macromolecules are only able tocross a nuclear pore if they are associated with a transport molecule(karyopherin) that temporarily interacts with a nucleoporin's FG-repeatsegments. FG-nucleoporins also contain a globular portion that serves asan anchor for attachment to the nuclear pore complex.

Karyopherins and their cargo are passed between FG-repeats until theydiffuse down their concentration gradient and through the nuclear porecomplex. The release of their cargo from karyopherins is driven by Ran,a G protein. Ran is small enough that it can diffuse through nuclearpores down its concentration gradient without interacting withnucleoporins. Ran binds to either GTP or GDP and has the ability tochange a karyopherin's affinity for its cargo. Inside the nucleus,RanGTP causes an importin karyopherin to change conformation, allowingits cargo to be released. RanGTP can also bind to exportin karyopherinsand pass through the nuclear pore. Once it has reached the cytosol,RanGTP can be hydrolyzed to RanGDP, allowing the exportin's cargo to bereleased.

Adapting artificially engineered protein polymers from consensus repeatsof natural proteins is an attractive approach to mimic the unprecedentedperformance of natural materials. Tough silk-like polypeptides,thermoresponsive elastin-like polypeptides, and resilient and elasticresilin-like polypeptides have been synthesized to mimic the functionsof natural materials. Important design principles have been developedfor these artificial biopolymers that enable rational control over theirthermodynamic, structural, and mechanical properties. The simplifiedrepeat allows for a detailed understanding ofsequence-structure-property relationships to be developed, and thesetailor-made materials open up opportunities for applications such asdrug delivery, tissue engineering, photonic films and smart responsivedevices.

An additional natural material that has interesting engineeringproperties is the protein matrix which fills the nuclear pore complex(NPC) in the nuclear envelope and controls transport into the nucleus.It allows passage of less than 0.1% of all proteins while translocatingover 1,000 molecules per pore per second. Ribbeck K et al., EMBO J 20:1320 (2001); Yang W D et al., Proc Natl Acad Sci USA 101: 12887 (2004).The protein matrix is composed of nucleoporins, proteins containingPhe-Gly (FG) repeat sequences which contribute to specific binding ofthe nuclear transport receptors (NTRs) that facilitate transport of aspecific subset of biological molecules into the matrix.

Individual nucleoporins can form hydrogels in vitro that recapitulatethe enhanced permeability of selectively-labeled macromolecules into thegel, similar to the intact NPC, with varying degrees of passivediffusion of inert molecules. Labokha A A et al., EMBO J 32: 204 (2013);Jovanovic-Talisman T et al., Nature 457: 1023 (2009). This selectivityis rare in synthetic polymer hydrogels, making these natural materialsan intriguing model for new filtration technologies. In spite of theadvanced filtering function of natural nucleoporin hydrogels, until nowa fundamental understanding of the sequence-structure-propertyrelationships needed for materials engineering has been lacking, due tothe complex sequence of the proteins and the inability to synthesizethem recombinantly in high yields.

SUMMARY OF THE INVENTION

To adapt the function of nucleoporin hydrogels in a biosyntheticmaterial, artificially engineered protein polymers were designed thatcan replicate the biological selective transport of the hydrogel in asynthetic mimic using a consensus repeat adapted from awell-investigated nucleoporin, Nsp1. Frey S et al., Science 314: 815(2006); Ader C et al., Proc Natl Acad Sci USA 107: 6281 (2010); Frey Set al., Cell 130: 512 (2007). The polymers provide a valuable tool formaterial engineering and an opportunity to tune the selectivity,transport rates, and barrier function of nucleoporin-inspired materialsthrough rational repeat sequence design.

As described in detail herein, designed peptides 1NLP and 2NLP,extracted from partial NspI nucleoporin, are useful for the preparationof nucleoporin-based hydrogels characterized by selective filteringcapability.

As described herein, recombinant nucleoporin-like polypeptides P-1NLP-P,P-2NLP-P, and P-cNspI-P are useful for the preparation of hydrogelscharacterized by selective filtering capability.

As described in detail herein, hydrogels of the invention are useful asselectively permeable barriers.

Also as described in detail herein, hydrogels of the invention areuseful for sequestration of compounds, including macromolecules.

Additionally, hydrogels of the invention find use as models for thenuclear pore in assays for nuclear permeability of drugs, biomaterials,nanoparticles, and other compounds.

As described in detail herein, various nuclear transport receptors, suchas importin β and NTF2, can be used as carriers which can selectivelybring target molecules into P-1NLP-P, P-2NLP-P, and P-cNspI-P hydrogels.

Also as described in detail herein, hydrogels of the invention areuseful for collecting selected target molecules into hydrogel withnuclear transport receptor associated with target molecule-specificbinding tag.

An aspect of the invention is a polypeptide comprising a plurality ofcontiguous instances of a subsequence represented by PAFSFGAKPDEKKDSDTSK(SEQ ID NO:1).

In certain embodiments, the polypeptide comprises 16 contiguousinstances of the subsequence represented by SEQ ID NO:1.

In certain embodiments, the polypeptide consists of 16 contiguousinstances of the subsequence represented by SEQ ID NO:1.

In certain embodiments, the polypeptide further comprises a firstleucine zipper domain endblock flanking the N-terminal end of theplurality of contiguous instances of the subsequence represented by SEQID NO:1; and a second leucine zipper domain endblock flanking theC-terminal end of the plurality of contiguous instances of thesubsequence represented by SEQ ID NO:1.

In certain embodiments, the leucine zipper domain endblock consists of aP domain.

In certain embodiments, the P domain consists of the peptide representedby APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the polypeptide comprises the sequencerepresented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMES DAS (SEQ IDNO:4).

An aspect of the invention is a polypeptide comprising a plurality ofcontiguous instances of a subsequence represented by PAFSFGAKPDEKKDDDTSK(SEQ ID NO:2).

In certain embodiments, the polypeptide comprises 16 contiguousinstances of the subsequence represented by SEQ ID NO:2.

In certain embodiments, the polypeptide consists of 16 contiguousinstances of the subsequence represented by SEQ ID NO:2.

In certain embodiments, the polypeptide further comprises a firstleucine zipper domain endblock flanking the N-terminal end of theplurality of contiguous instances of the subsequence represented by SEQID NO:2; and a second leucine zipper domain endblock flanking theC-terminal end of the plurality of contiguous instances of thesubsequence represented by SEQ ID NO:2.

In certain embodiments, the leucine zipper domain endblock consists of aP domain.

In certain embodiments, the P domain consists of the peptide representedby APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the polypeptide comprises the sequencerepresented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVM ESDAS (SEQ IDNO:5).

An aspect of the invention is a polypeptide comprising a core sequencerepresented by PSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSK (SEQ ID NO:6); and a first leucine zipperdomain endblock flanking the N-terminal end or the C-terminal end of thecore sequence.

In certain embodiments, the first leucine zipper domain endblock flanksthe N-terminal end of the core sequence.

In certain embodiments, the polypeptide further comprises a secondzipper domain endblock flanking the C-terminal end of the core sequence.

In certain embodiments, the leucine zipper domain endblock consists of aP domain.

In certain embodiments, the P domain consists of the peptide representedby APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the polypeptide comprises the sequencerepresented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSKPAFTFGTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:7).

An aspect of the invention is a nucleic acid molecule encoding apolypeptide of the invention.

An aspect of the invention is an expression vector comprising a nucleicacid molecule of the invention.

An aspect of the invention is a cell, comprising an expression vector ofthe invention.

An aspect of the invention is a hydrogel, comprising a polypeptide ofthe invention.

An aspect of the invention is a filtering device, comprising a hydrogelof the invention; and a housing or support for the hydrogel.

An aspect of the invention is a drug delivery device, comprising a drug;and a hydrogel of the invention.

An aspect of the invention is a method of separating or selectivelyfiltering macromolecules, comprising contacting a source ofmacromolecules with a hydrogel of the invention.

In certain embodiments, the macromolecule is selected from the groupconsisting of RNA, mRNA, DNA, proteins, glycoproteins, carbohydrates,lipids, toxins, and any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts design of synthetic protein polymers, P-cNsp1-P andP-NLPs-P, which gel by association of pentameric (P) coiled-coilendblock domains (coils). Filled circles represent Phe-Gly (FG)sequences.

FIG. 1B depicts 3D gel network formed from assembly of designedartificially engineered proteins. Black dotted circles highlightPhe-mediated interactions within synthetic hydrogels.

FIG. 2A depicts a series of three western blots SDS-PAGE of indicatedlyophilized protein samples.

FIG. 2B is a graph depicting yields of designed proteins flanked by Pdomains.

FIG. 3A is a panel of three western blots depicting expression levels of1NLP, 2NLP, and cNsp1*, where cNsp1* was obtained as a product fromP-intein-cNsp1.

FIG. 3B is a bar graph depicting expression levels of cNsp1, 1NLP, 2NLP,and cNsp1*, where cNsp1* was obtained as a product from P-intein-cNsp1.

FIG. 4A depicts a schematic of capillary transport assay set-up. Blue(darker) and green (lighter) circles represent importin β andIBB-MBP-EGFP, respectively.

FIG. 4B depicts a time course transport measurement of 20 w/v %P-cNsp1-P hydrogel with β.

FIG. 4C is a graph depicting 20 w/v % P-cNsp1-P hydrogels in thepresence (solid line) or absence (dotted line) of importin β.

FIG. 4D is a graph depicting 20 w/v % P-1NLP-P hydrogels in the presence(solid line) or absence (dotted line) of importin β. Scale bar, 900 μm.

FIG. 4E is a graph depicting 20 w/v % P-2NLP-P hydrogels in the presence(solid line) or absence (dotted line) of importin β. Scale bar, 900 μm.

FIG. 4F is a graph depicting absorption of cargo-importin β complexes byP-cNsp1-P, P-1NLP-P and P-2NLP-P hydrogels in one hour. * denotesp<0.05.

FIG. 4G is a graph depicting fluorescence intensity measurements onP-2NLP-P hydrogels (20 w/v %) with 10% 1,6 hexanediol. Scale bar, 900μm.

FIG. 4H is a graph depicting selective permeability test performed onP-1NLP-P biosynthetic hydrogels (20 w/v %) with the addition of 5 μMMBP-mCherry, a model inert molecule, into 5 μM IBB-MBP-EGFP/importin βcargo complex mixtures. Scale bar, 900 μm.

FIG. 4I is a graph depicting selective permeability test performed onP-2NLP-P biosynthetic hydrogels (20 w/v %) with the addition of 5 μMMBP-mCherry, a model inert molecule, into 5 μM IBB-MBP-EGFP/importin βcargo complex mixtures. Scale bar, 900 μm.

FIG. 5A is a graph depicting selective permeability of P-2NLP-P gel in20 w/v % FIG. 5B is a graph depicting selective permeability of P-2NLP-Pgel in 10 w/v %.

FIG. 6 is a panel of four western blots depicting protein expressionlevels of Nsp1, P-cNsp1-P, P-1NLP-P, and P-2NLP-P. L, protein ladder; E,elution fraction.

FIG. 7A is a graph depicting frequency sweep, linear oscillatory shearrheology of 20 w/v % P-cNsp1-P hydrogels in the absence (blue curves) orpresence (red curves) of 10% 1,6 hexanediol. The gel modulus and thecrossover frequency in the absence of hexanediol are 9.3 kPa and 0.08rad/s.

FIG. 7B is a graph depicting frequency sweep, linear oscillatory shearrheology of 20 w/v % P-1NLP-P hydrogels in the absence (blue curves) orpresence (red curves) of 10% 1,6 hexanediol. The gel modulus and thecrossover frequency in the absence of hexanediol are 10.7 kPa and 0.02rad/s.

FIG. 7C is a graph depicting frequency sweep, linear oscillatory shearrheology of 20 w/v % P-2NLP-P hydrogels in the absence (blue curves) orpresence (red curves) of 10% 1,6 hexanediol. The gel modulus and thecrossover frequency in the absence of hexanediol are 7.5 kPa and 0.04rad/s.

FIG. 7D is a graph depicting frequency sweep, linear oscillatory shearrheology of 20 w/v % P-C₃₀-P gel, which lacks FG repeats in itsmidblock. C is a peptide having amino acid sequence AGAGAGPEG (SEQ IDNO:8).

FIG. 7E is a graph depicting Raman spectra of 20 w/v % cNsp1 midblocks,measured in buffer containing 50 mM Tris/HCl (pH 7.5) and 200 mM NaCl(blue curve) and with the addition of 10% hexanediol (red curve). Theshaded boxes highlight Raman bands of 486, 685 and 710 cm⁻¹ thatdecrease in intensity with the addition of hexanediol.

FIG. 7F is a graph depicting Raman spectra of 20 w/v % 1NLP midblocks,measured in buffer containing 50 mM Tris/HCl (pH 7.5) and 200 mM NaCl(blue curve) and with the addition of 10% hexanediol (red curve). Theshaded boxes highlight Raman bands of 486, 685 and 710 cm⁻¹ thatdecrease in intensity with the addition of hexanediol.

FIG. 7G is a graph depicting Raman spectra of 20 w/v % 2NLP midblocks,measured in buffer containing 50 mM Tris/HCl (pH 7.5) and 200 mM NaCl(blue curve) and with the addition of 10% hexanediol (red curve). Theshaded boxes highlight Raman bands of 486, 685 and 710 cm⁻¹ thatdecrease in intensity with the addition of hexanediol.

FIG. 8 is a series of four graphs depicting strain sweep oscillatoryshear rheology of indicated 20 w/v % hydrogels at 100 rad/s and 25° C. Cis a peptide having amino acid sequence AGAGAGPEG (SEQ ID NO:8).

FIG. 9A is a western blot depicting P-C₃₀-P. C is a peptide having aminoacid sequence AGAGAGPEG (SEQ ID NO:8).

FIG. 9B is a graph depicting strain sweep oscillatory shear rheology ofindicated P-C₃₀-P hydrogels prepared by hydrating lyophilized samples(20 w/v %) with buffer containing 50 mM Tris/HCl (pH 7.5) and 200 mMNaCl in the presence or absence of 10% 1,6-hexanediol.

FIG. 10 is a graph depicting Raman spectra of cNsp1 (20 w/v %) withselected band assignments; buffer contained 50 mM Tris/HCl (pH 7.5) and200 mM NaCl. Δ=deformation; σ=stretching.

FIG. 11 is a graph depicting Raman spectra of cNsp1 (upper spectrum) and1NLP (lower spectrum) lyophilized samples with selected bandassignments.

FIG. 12 is a series of three graphs depicting diffusion of various sizesof FITC-dextran through the indicated hydrogels. Gel pore radii areestimated between 2.3 nm and 4.5 nm.

FIG. 13A is a graph depicting permeability profile of the P-cNsp1-Phydrogel for various sizes of FITC-dextran (as per FIG. 12). Theflorescence intensity profile on the gel at 1 hour is shown. Areas underthe solid curves (>0 μm) were calculated and compared to the passivediffusion by inert molecules (dashed curve).

FIG. 13B is a graph depicting permeability profile of the P-1NLP-Phydrogel for various sizes of FITC-dextran (as per FIG. 12). Theflorescence intensity profile on the gel at 1 hour is shown. Areas underthe solid curves (>0 μm) were calculated and compared to the passivediffusion by inert molecules (dashed curve).

FIG. 13C is a graph depicting permeability profile of the P-2NLP-Phydrogel for various sizes of FITC-dextran (as per FIG. 12). Theflorescence intensity profile on the gel at 1 hour is shown. Areas underthe solid curves (>0 μm) were calculated and compared to the passivediffusion by inert molecules (dashed curve).

FIG. 14A is a graph depicting indicated gel and buffer interface changesby gel swelling during time lapse measurement of the capillary assay forIBB-MBP-GFP.

FIG. 14B is a graph depicting indicated gel and buffer interface changesby gel swelling during time lapse measurement of the capillary assay forIBB-MBP-GFP+importin β.

FIG. 15 is a graph depicting circular dichroism (CD) analysis of cNsp1and indicated NLPs.

FIG. 16 is a graph depicting permeability profiles of P-cNsp1-P hydrogel(20 w/v %) with 10% 1,6 hexanediol, for IBB-MBP-GFP with or withoutimportin β.

FIG. 17A is a graph depicting time lapse measurements of fluorescenceprofiles measured to investigate the selective permeability of P-2NLP-Pgel (20 w/v %) in the mixture of IBB-MBP-EGFP, MBP-mCherry, and β. Solidcurves represent green fluorescence intensity observed in the gel.

FIG. 17B is a graph depicting movement of the gel/buffer boundary overthe course of the selective permeability tests of P-2NLP-P gel (20 w/v%) in the mixture of IBB-MBP-EGFP, MBP-mCherry, and β. For the controlexperiment, only MBP-mCherry was tested.

FIG. 18 is a schematic depicting a method for selective capture of atarget molecule by a hydrogel of the invention.

FIG. 19A is a photographic image depicting NTF2-GFP in P-2NLP-P.

FIG. 19B is a photographic image depicting NTF2-GFP in P-1NLP-P.

FIG. 19C is a photographic image depicting NTF2-GFP in P-cNsp1-P.

FIG. 19D is a photographic image depicting 40 kg/mol FITC-dextran inP-2NLP-P.

FIG. 19E is a photographic image depicting 40 kg/mol FITC-dextran inP-1NLP-P.

FIG. 19F is a photographic image depicting 40 kg/mol FITC-dextran inP-cNsp1-P.

DETAILED DESCRIPTION OF THE INVENTION

Recent in vitro results indicate that the recombinant Nsp1²⁻⁶⁰¹ can bedivided into an N-terminal sequence Nsp1²⁻²⁷⁷ and a C-terminal sequenceNsp1²⁷⁴⁻⁶⁰¹. Ader C et al., Proc Natl Acad Sci USA 107: 6281 (2010). InNsp1, the C-terminal sequence contributes to selective transport ofNTR-cargo complexes and less non-specific binding of inert molecules,core functions for selective transport. However, the C-terminal sequencealone forms a liquid that cannot restrict the passage of inertmolecules. The N-terminal sequence is critical for gelation, suggestingthat network formation is required for a fully functional selectivetransport system.

To prepare synthetic gels, the N-terminal sequence of Nsp1, which gelsslowly over a period of hours, was replaced with well-investigatedpentameric (P) coiled-coil domains flanking the C-terminal sequence(cNsp1). Petka W A et al., Science 281: 389 (1998); Shen W et al., NatMater 5: 153 (2006); Olsen B D et al., Macromolecules 43: 9094 (2010).This triblock protein construct, P-cNsp1-P, gels in minutes, and thetransient interactions of the P domains allow network relaxation that isthought to be critical to transport. Ribbeck K et al., EMBO J 20: 1320(2001).

Analysis of the cNsp1 consensus sequence (Nsp1²⁸²⁻⁵⁸⁵) allows reductionof the protein to a polymer of short repeating segments. Nsp1²⁸²⁻⁵⁸⁵ iscomposed of 16 repeats of a 19-amino acid Phe-Gly (FG)-containingsequence, with a high consensus at each position except position 15,where equal numbers of Asp (D) and Ser (S) are observed. To elucidatethe 16 consecutive 19-amino acid segments, the 304-amino acid sequenceof Nsp1²⁸²⁻⁵⁸⁵ (SEQ ID NO:9) can be written thus:

PSFSFGAKSDENKAGATSK PAFSFGAKPEEKKDDNSSK PAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSK PAFSFGAKSDEKKDGDASK PAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSK PAFSFGAKSNEDKQDGTAK PAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASK PAFSFGAKSDEKKDSDSSK PAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSK PAFSFGAKANEKKESDESK SAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSKwhere the repeating FG sequences and the D and S residues at position 15are shown in bold.

To capture the highest frequency of occurrence in all positions ofcNsp1, two separate repeat units were designed: one where position 15was Asp (D) (PAFSFGAKPDEKKDDDTSK; SEQ ID NO:2), and another whereposition 15 was Ser (S) (PAFSFGAKPDEKKDSDTSK; SEQ ID NO:1). Thesesequences were cloned to form an artificial protein polymer of 16 suchunits, producing two nucleoporin-like polypeptides (NLPs) denoted 1NLPand 2NLP, respectively. Both NLPs were genetically fused with P domainendblocks (P-1NLP-P and P-2NLP-P, FIG. 1A) to construct polymers thatform gels due to coiled-coil physical association (FIG. 1B). Since thesesimplified NLP polymers can mimic the properties of natural cNsp1, thepolymers represent a valuable tool for material engineering and anopportunity to tune the selectivity, transport rates and barrierfunction of nucleoporin-inspired materials through rational repeatsequence design.

Compounds of the Invention

2NLP

An aspect of the invention is a polypeptide comprising a plurality ofcontiguous instances of a subsequence represented by PAFSFGAKPDEKKDSDTSK(SEQ ID NO:1).

In certain embodiments, the polypeptide consists of a plurality ofcontiguous instances of a subsequence represented by SEQ ID NO:1.

In certain embodiments, the polypeptide comprises 16 contiguousinstances of the subsequence represented by SEQ ID NO:1.

In certain embodiments, the polypeptide consists of 16 contiguousinstances of the subsequence represented by SEQ ID NO:1.

In certain embodiments, the polypeptide further comprises a firstleucine zipper domain endblock flanking the N-terminal end of theplurality of contiguous instances of the subsequence represented by SEQID NO:1.

In certain embodiments, the polypeptide further comprises a firstleucine zipper domain endblock flanking the C-terminal end of theplurality of contiguous instances of the subsequence represented by SEQID NO:1.

In certain embodiments, the first leucine zipper domain endblockcomprises a pentameric coiled-coil domain (P domain).

In certain embodiments, the first leucine zipper domain endblockconsists of a pentameric coiled-coil domain (P domain).

In certain embodiments, the P domain comprises the peptide representedby APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the P domain consists of the peptide representedby APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the polypeptide further comprises a firstleucine zipper domain endblock flanking the N-terminal end of theplurality of contiguous instances of the subsequence represented by SEQID NO:1; and a second leucine zipper domain endblock flanking theC-terminal end of the plurality of contiguous instances of thesubsequence represented by SEQ ID NO:1.

In certain embodiments, the first leucine zipper domain endblockcomprises a P domain.

In certain embodiments, the first leucine zipper domain endblockconsists of a P domain.

In certain embodiments, the first leucine zipper domain endblock Pdomain comprises the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the first leucine zipper domain endblock Pdomain consists of the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the second leucine zipper domain endblockcomprises a P domain.

In certain embodiments, the second leucine zipper domain endblockconsists of a P domain.

In certain embodiments, the second leucine zipper domain endblock Pdomain comprises the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the second leucine zipper domain endblock Pdomain consists of the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the first leucine zipper domain endblockcomprises a P domain; and the second leucine zipper domain endblockcomprises a P domain.

In certain embodiments, the first leucine zipper domain endblockcomprises a P domain; and the second leucine zipper domain endblockconsists of a P domain.

In certain embodiments, the first leucine zipper domain endblockconsists of a P domain; and the second leucine zipper domain endblockcomprises a P domain.

In certain embodiments, the first leucine zipper domain endblockconsists of a P domain; and the second leucine zipper domain endblockconsists of a P domain.

In certain embodiments, the first leucine zipper domain endblock Pdomain comprises the peptide represented by SEQ ID NO:3; and the secondleucine zipper domain endblock P domain comprises the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the first leucine zipper domain endblock Pdomain comprises the peptide represented by SEQ ID NO:3; and the secondleucine zipper domain endblock P domain consists of the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the first leucine zipper domain endblock Pdomain consists of the peptide represented by SEQ ID NO:3; and thesecond leucine zipper domain endblock P domain comprises the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the first leucine zipper domain endblock Pdomain consists of the peptide represented by SEQ ID NO:3; and thesecond leucine zipper domain endblock P domain consists of the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the polypeptide comprises the sequencerepresented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMES DAS (SEQ IDNO:4).

In certain embodiments, the polypeptide consists of the sequencerepresented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMES DAS (SEQ IDNO:4).

1NLP

An aspect of the invention is a polypeptide comprising a plurality ofcontiguous instances of a subsequence represented by PAFSFGAKPDEKKDDDTSK(SEQ ID NO:2).

In certain embodiments, the polypeptide consists of a plurality ofcontiguous instances of a subsequence represented by SEQ ID NO:2.

In certain embodiments, the polypeptide comprises 16 contiguousinstances of the subsequence represented by SEQ ID NO:2.

In certain embodiments, the polypeptide consists of 16 contiguousinstances of the subsequence represented by SEQ ID NO:2.

In certain embodiments, the polypeptide further comprises a firstleucine zipper domain endblock flanking the N-terminal end of theplurality of contiguous instances of the subsequence represented by SEQID NO:2.

In certain embodiments, the polypeptide further comprises a firstleucine zipper domain endblock flanking the C-terminal end of theplurality of contiguous instances of the subsequence represented by SEQID NO:2.

In certain embodiments, the first leucine zipper domain endblockcomprises a pentameric coiled-coil domain (P domain).

In certain embodiments, the first leucine zipper domain endblockconsists of a pentameric coiled-coil domain (P domain).

In certain embodiments, the P domain comprises the peptide representedby APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the P domain consists of the peptide representedby APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the polypeptide further comprises a firstleucine zipper domain endblock flanking the N-terminal end of theplurality of contiguous instances of the subsequence represented by SEQID NO:2; and a second leucine zipper domain endblock flanking theC-terminal end of the plurality of contiguous instances of thesubsequence represented by SEQ ID NO:2.

In certain embodiments, the first leucine zipper domain endblockcomprises a P domain.

In certain embodiments, the first leucine zipper domain endblockconsists of a P domain.

In certain embodiments, the first leucine zipper domain endblock Pdomain comprises the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the first leucine zipper domain endblock Pdomain consists of the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the second leucine zipper domain endblockcomprises a P domain.

In certain embodiments, the second leucine zipper domain endblockconsists of a P domain.

In certain embodiments, the second leucine zipper domain endblock Pdomain comprises the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the second leucine zipper domain endblock Pdomain consists of the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the first leucine zipper domain endblockcomprises a P domain; and the second leucine zipper domain endblockcomprises a P domain.

In certain embodiments, the first leucine zipper domain endblockcomprises a P domain; and the second leucine zipper domain endblockconsists of a P domain.

In certain embodiments, the first leucine zipper domain endblockconsists of a P domain; and the second leucine zipper domain endblockcomprises a P domain.

In certain embodiments, the first leucine zipper domain endblockconsists of a P domain; and the second leucine zipper domain endblockconsists of a P domain.

In certain embodiments, the first leucine zipper domain endblock Pdomain comprises the peptide represented by SEQ ID NO:3; and the secondleucine zipper domain endblock P domain comprises the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the first leucine zipper domain endblock Pdomain comprises the peptide represented by SEQ ID NO:3; and the secondleucine zipper domain endblock P domain consists of the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the first leucine zipper domain endblock Pdomain consists of the peptide represented by SEQ ID NO:3; and thesecond leucine zipper domain endblock P domain comprises the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the first leucine zipper domain endblock Pdomain consists of the peptide represented by SEQ ID NO:3; and thesecond leucine zipper domain endblock P domain consists of the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the polypeptide comprises the sequencerepresented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVM ESDAS (SEQ IDNO:5).

In certain embodiments, the polypeptide consists of the sequencerepresented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVM ESDAS (SEQ IDNO:5).

cNsp1

An aspect of the invention is a polypeptide comprising a core sequencerepresented by PSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSK (SEQ ID NO:6); and a first leucine zipperdomain endblock flanking the N-terminal end or the C-terminal end of thecore sequence.

In certain embodiments, the polypeptide consists of a core sequencerepresented by PSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSK (SEQ ID NO:6); and a first leucine zipperdomain endblock flanking the N-terminal end or the C-terminal end of thecore sequence.

In certain embodiments, the first leucine zipper domain endblock flanksthe N-terminal end of the core sequence.

In certain embodiments, the first leucine zipper domain endblock flanksthe C-terminal end of the core sequence.

In certain embodiments, the first leucine zipper domain endblockcomprises a pentameric coiled-coil domain (P domain).

In certain embodiments, the first leucine zipper domain endblockconsists of a pentameric coiled-coil domain (P domain).

In certain embodiments, the first leucine zipper domain endblock Pdomain comprises the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the first leucine zipper domain endblock Pdomain consists of the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).

In certain embodiments, the polypeptide comprises the core domain, thefirst leucine zipper domain endblock flanking the N-terminal end of thecore sequence, and a second leucine zipper domain endblock flanking theC-terminal end of the core sequence.

In certain embodiments, the polypeptide consists of the core domain, thefirst leucine zipper domain endblock flanking the N-terminal end of thecore sequence, and a second leucine zipper domain endblock flanking theC-terminal end of the core sequence.

In certain embodiments, the first leucine zipper domain endblock Pdomain comprises the peptide represented by SEQ ID NO:3; and the secondleucine zipper domain endblock P domain comprises the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the first leucine zipper domain endblock Pdomain comprises the peptide represented by SEQ ID NO:3; and the secondleucine zipper domain endblock P domain consists of the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the first leucine zipper domain endblock Pdomain consists of the peptide represented by SEQ ID NO:3; and thesecond leucine zipper domain endblock P domain comprises the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the first leucine zipper domain endblock Pdomain consists of the peptide represented by SEQ ID NO:3; and thesecond leucine zipper domain endblock P domain consists of the peptiderepresented by SEQ ID NO:3.

In certain embodiments, the polypeptide comprises the sequencerepresented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSKPAFTFGTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:7).

In certain embodiments, the polypeptide consists of the sequencerepresented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSKPAFTFGTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:7).

An aspect of the invention is a nucleic acid molecule encoding apolypeptide of the invention.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:4. For example, in one embodiment, a DNAsequence encoding SEQ ID NO:4 is:

(SEQ ID NO: 18) gcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggcgcgagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaaccagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaaccagcgcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagc.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:5. For example, in one embodiment, a DNAsequence encoding SEQ ID NO:5 is:

(SEQ ID NO: 19) gcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggcgcgagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaaccagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaaccagcgcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagc.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:6. For example, in one embodiment, a DNAsequence encoding SEQ ID NO:6 is:

(SEQ ID NO: 20) ccgagctttagctttggcgcgaaaagcgatgaaaacaaagcgggcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaccggatgaaaacaaagcgagcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatagcgatagcagcaaaccggcgtttagctttggcaccaaaagcaacgaaaaaaaagatagcggcagcagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaaaacgatgaagtgagcaaaccggcgtttagctttggcgcgaaagcgaacgaaaaaaaagaaagcgatgaaagcaaaagcgcgtttagctttggcagcaaaccgaccggcaaagaagaaggcgatggcgcgaaagcggcgattagctttggcgcgaaaccggaagaacagaaaagcagc gataccagcaaa.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:7. For example, in one embodiment, a DNAsequence encoding SEQ ID NO: 7 is:

(SEQ ID NO: 21) gcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggcgcgagcgataacaaaaccaccaacaccaccccgagctttagctttggcgcgaaaagcgatgaaaacaaagcgggcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaccggatgaaaacaaagcgagcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatagcgatagcagcaaaccggcgtttagctttggcaccaaaagcaacgaaaaaaaagatagcggcagcagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaaaacgatgaagtgagcaaaccggcgtttagctttggcgcgaaagcgaacgaaaaaaaagaaagcgatgaaagcaaaagcgcgtttagctttggcagcaaaccgaccggcaaagaagaaggcgatggcgcgaaagcggcgattagctttggcgcgaaaccggaagaacagaaaagcagcgataccagcaaaccggcgtttacctttggcaccagcgcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagc.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:10 (see below). For example, in one embodiment,a DNA sequence encoding SEQ ID NO:10 is:

(SEQ ID NO: 22) atggatattggcattaacagcgatccgagcaccggcgcgggcgcgtttggcaccggccagagcacctttggctttaacaacagcgcgccgaacaacaccaacaacgcgaacagcagcattaccccggcgtttggcagcaacaacaccggcaacaccgcgtttggcaacagcaacccgaccagcaacgtgtttggcagcaacaacagcaccaccaacacctttggcagcaacagcgcgggcaccagcctgtttggcagcagcagcgcgcagcagaccaaaagcaacggcaccgcgggcggcaacacctttggcagcagcagcctgtttaacaacagcaccaacagcaacaccaccaaaccggcgtttggcggcctgaactttggcggcggcaacaacaccaccccgagcagcaccggcaacgcgaacaccagcaacaacctgtttggcgcgaccgcgaacgcgaacaaaccggcgtttagctttggcgcgaccaccaacgatgataaaaaaaccgaaccggataaaccggcgtttagctttaacagcagcgtgggcaacaaaaccgatgcgcaggcgccgaccaccggctttagctttggcagccagctgggcggcaacaaaaccgtgaacgaagcggcgaaaccgagcctgagctttggcagcggcagcgcgggcgcgaacccggcgggcgcgagccagccggaaccgaccaccaacgaaccggcgaaaccggcgctgagctttggcaccgcgaccagcgataacaaaaccaccaacaccaccccgagctttagctttggcgcgaaaagcgatgaaaacaaagcgggcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaccggatgaaaacaaagcgagcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatagcgatagcagcaaaccggcgtttagctttggcaccaaaagcaacgaaaaaaaagatagcggcagcagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaaaacgatgaagtgagcaaaccggcgtttagctttggcgcgaaagcgaacgaaaaaaaagaaagcgatgaaagcaaaagcgcgtttagctttggcagcaaaccgaccggcaaagaagaaggcgatggcgcgaaagcggcgattagctttggcgcgaaaccggaagaacagaaaagcagcgataccagcaaaccggcgtttacctttggcaaactggcggcggcgctggaacatcatcatcatcatc at.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:11 (see below). For example, in one embodiment,a DNA sequence encoding SEQ ID NO:11 is:

(SEQ ID NO: 23) atggatattggcattaacagcgatccgggcagcggcagcggcgcgagcgataacaaaaccaccaacaccaccccgagctttagctttggcgcgaaaagcgatgaaaacaaagcgggcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaccggatgaaaacaaagcgagcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagattggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagattggcgcgaaaagcgatgaaaaaaaagatagcgatagcagcaaaccggcgtttagattggcaccaaaagcaacgaaaaaaaagatagcggcagcagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaaaacgatgaagtgagcaaaccggcgtttagctttggcgcgaaagcgaacgaaaaaaaagaaagcgatgaaagcaaaagcgcgtttagctttggcagcaaaccgaccggcaaagaagaaggcgatggcgcgaaagcggcgattagattggcgcgaaaccggaagaacagaaaagcagcgataccagcaaaccggcgtttacctttggcaccagcggcagcggcaaactggcggcggcgctggaacatcatcatcatcatcat.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:12 (see below). For example, in one embodiment,a DNA sequence encoding SEQ ID NO:12 is:

(SEQ ID NO: 24) atggatattggcattaacagcgatccggcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggcgcgagcgcgattagcggcgatagcctgattagcctggcgagcaccggcaaacgcgtgagcattaaagatctgctggatgaaaaagattttgaaatttgggcgattaacgaacagaccatgaaactggaaagcgcgaaagtgagccgcgtgttttgcaccggcaaaaaactggtgtatattctgaaaacccgcctgggccgcaccattaaagcgaccgcgaaccatcgctttctgaccattgatggctggaaacgcctggatgaactgagcctgaaagaacatattgcgctgccgcgcaaactggaaagcagcagcctgcagctgagcccggaaattgaaaaactgagccagagcgatatttattgggatagcattgtgagcattaccgaaaccggcgtggaagaagtgtttgatctgaccgtgccgggcccgcataactttgtggcgaacgatattattgtgcataacgcgagcgataacaaaaccaccaacaccaccccgagctttagctttggcgcgaaaagcgatgaaaacaaagcgggcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaccggatgaaaacaaagcgagcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatagcgatagcagcaaaccggcgtttagctttggcaccaaaagcaacgaaaaaaaagatagcggcagcagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaaaacgatgaagtgagcaaaccggcgtttagctttggcgcgaaagcgaacgaaaaaaaagaaagcgatgaaagcaaaagcgcgtttagctttggcagcaaaccgaccggcaaagaagaaggcgatggcgcgaaagcggcgattagctttggcgcgaaaccggaagaacagaaaagcagcgataccagcaaaccggcgtttacctttggcaccagcggcagcggcaaactggcggcggcgctggaacatcatcatcatcatcat.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:13 (see below). For example, in one embodiment,a DNA sequence encoding SEQ ID NO:13 is:

(SEQ ID NO: 25) atggatattggcattaacagcgatccgggcagcggcgcgagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaaccagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaaccagcggcagcggcaaactggcggcggcgctggaacatc atcatcatcatcat.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:14 (see below). For example, in one embodiment,a DNA sequence encoding SEQ ID NO:14 is:

(SEQ ID NO: 26) atggatattggcattaacagcgatccgggcagcggcgcgagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaaccagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagattggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaaccagcaaaaccagcggcagcggcaaactggcggcggcgctggaacatcatcatcatcatcat.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:15 (see below). For example, in one embodiment,a DNA sequence encoding SEQ ID NO:15 is:

(SEQ ID NO: 27) atggatattggcattaacagcgatccggcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggcgcgagcgataacaaaaccaccaacaccaccccgagctttagctttggcgcgaaaagcgatgaaaacaaagcgggcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaccggatgaaaacaaagcgagcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagattggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatagcgatagcagcaaaccggcgtttagctttggcaccaaaagcaacgaaaaaaaagatagcggcagcagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaaaacgatgaagtgagcaaaccggcgtttagctttggcgcgaaagcgaacgaaaaaaaagaaagcgatgaaagcaaaagcgcgtttagctttggcagcaaaccgaccggcaaagaagaaggcgatggcgcgaaagcggcgattagctttggcgcgaaaccggaagaacagaaaagcagcgataccagcaaaccggcgtttacctttggcaccagcgcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggcaaactggcggcggcgctggaacatcatcatcatcatcat.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:16 (see below). For example, in one embodiment,a DNA sequence encoding SEQ ID NO:16 is:

(SEQ ID NO: 28) atggatattggcattaacagcgatccggcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggcgcgagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaaccagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaaccagcgcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggcggcaaactggcggcggcgctggaacatcatcatca tcatcat.

In one embodiment, the nucleic acid molecule encodes a polypeptiderepresented by SEQ ID NO:17 (see below). For example, in one embodiment,a DNA sequence encoding SEQ ID NO:17 is:

(SEQ ID NO: 29) atggatattggcattaacagcgatccggcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggcgcgagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaaccagcccggcgtttagattggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagattggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaaccagcgcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggcggcaaactggcggcggcgctggaacatcatcatcatc atcat.

An aspect of the invention is an expression vector comprising a nucleicacid molecule of the invention.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a plasmid, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, namely expressionvectors, are capable of directing the expression of genes to which theyare operably linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the present invention is intended to include such other formsof expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Methods in Enzymology: GeneExpression Technology vol. 185, Academic Press, San Diego, Calif.(1991). Regulatory sequences include those which direct constitutiveexpression of a nucleotide sequence in many types of host cell and thosewhich direct expression of the nucleotide sequence only in certain hostcells (e.g., tissue-specific regulatory sequences). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or peptides, including proteinsor polypeptides, encoded by nucleic acids as described herein.

The recombinant expression vectors for use in the invention can bedesigned for expression of a polypeptide of the invention in prokaryotic(e.g., E. coli) or eukaryotic cells (e.g., insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells).Suitable host cells are discussed further in Goeddel, supra.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studieret al., p. 60-89, In Gene Expression Technology: Methods in Enzymologyvol. 185, Academic Press, San Diego, Calif., 1991). Target biomarkernucleic acid expression from the pTrc vector relies on host RNApolymerase transcription from a hybrid trp-lac fusion promoter. Targetbiomarker nucleic acid expression from the pET 11d vector relies ontranscription from a T7 gn10-lac fusion promoter mediated by aco-expressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacterium with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, p. 119-128,In Gene Expression Technology: Methods in Enzymology vol. 185, AcademicPress, San Diego, Calif., 1990). Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al., 1992, NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987,Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840)and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus, andSimian Virus 40 (SV40). For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen andBaltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985,Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss,1990, Science 249:374-379) and the α-fetoprotein promoter (Camper andTilghman, 1989, Genes Dev. 3:537-546).

In certain embodiments, the expression vector is a prokaryoticexpression vector.

In certain embodiments, the expression vector is a eukaryotic expressionvector.

An aspect of the invention is a cell comprising an expression vector ofthe invention.

In certain embodiments, the expression vector is a prokaryoticexpression vector, and the cell is a prokaryotic cell, e.g., E. coli.

In certain embodiments, the expression vector is a eukaryotic expressionvector, and the cell is a eukaryotic cell, e.g., a yeast cell, an insectcell, or a mammalian cell.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

Compositions of the Invention

An aspect of the invention is a hydrogel, comprising a polypeptide ofthe invention.

An aspect of the invention is a filtering device, comprising a hydrogelof the invention; and a housing or support for the hydrogel. In certainembodiments, the hydrogel is disposed within a housing. In certainembodiments, the hydrogel is disposed within and is in contact with thehousing. The housing can be configured in any suitable manner for theintended use of the filtering device, e.g., as a plate, a cone, amanifold, a cartridge, an open-ended tube, a closed-end tube (e.g., acentrifuge tube), etc. The housing optionally can include a fitting,e.g., a screw- or compression-Luer lock fitting, to facilitateconnection of the filtering device to a fluid path. In certainembodiments, the hydrogel is disposed upon a support. In certainembodiments, the hydrogel is disposed upon and is in contact with thesupport. The support can be configured in any suitable manner for theintended use of the filtering device, e.g., as a frame, a perforatedplate, a mesh, a fabric, a filter, etc.

In certain embodiments, the support, together with the hydrogel, can befitted or placed into a housing which is configured and arranged toreceive the support.

An aspect of the invention is a drug delivery device, comprising a drug;and a hydrogel of the invention.

In certain embodiments, the drug is a macromolecule.

In certain embodiments, the drug is a nucleic acid.

In certain embodiments, the drug is an RNA.

In certain embodiments, the drug is a DNA.

In certain embodiments, the drug is a polymer.

In certain embodiments, the drug is a polypeptide.

In certain embodiments, the drug is a protein.

In certain embodiments, the drug is a fusion protein.

In certain embodiments, the drug is an antibody or an antigen-bindingfragment of an antibody.

In certain embodiments, the drug is a cytokine.

In certain embodiments, the drug is a glycoprotein.

In certain embodiments, the drug is a carbohydrate.

In certain embodiments, the drug is a lipid.

In certain embodiments, the drug is a toxin.

In certain embodiments, the drug is a steroid.

In certain embodiments, the drug is a hormone, e.g., an estrogen or aprogestogen.

In certain embodiments, the drug is dispersed within the hydrogel.

In certain embodiments, the drug is effectively enveloped by thehydrogel. For example, the drug can be enclosed within a housing,wherein at least a portion of the housing is open to the environment butfor the presence of the hydrogel.

In certain embodiments, the drug is enveloped by the hydrogel. Forexample, the drug can be present as a core which is completelysurrounded by the hydrogel; the core can include more than a singleactive agent, and there can be one or more additional layers presentbetween the drug or core and the hydrogel. In certain embodiments, therecan be one or more additional layers external to the hydrogel.

Methods of the Invention

An aspect of the invention is a method of separating or selectivelyfiltering macromolecules, comprising contacting a source ofmacromolecules with a hydrogel of the invention. For example, a solutioncomprising a macromolecule can be contacted with a hydrogel of theinvention.

As used herein, the term “macromolecule” refers to any molecule having amolecular weight greater than about 1500 Da. In certain embodiments, amacromolecule has a molecular weight greater than or equal to about10,000 Da. In certain embodiments, a macromolecule has a molecularweight greater than or equal to about 20,000 Da. In certain embodiments,a macromolecule has a molecular weight greater than or equal to about30,000 Da. In certain embodiments, a macromolecule has a molecularweight greater than or equal to about 40,000 Da. In certain embodiments,a macromolecule has a molecular weight greater than or equal to about50,000 Da. In certain embodiments, a macromolecule has a molecularweight greater than or equal to about 60,000 Da. In certain embodiments,a macromolecule has a molecular weight greater than or equal to about70,000 Da. In certain embodiments, a macromolecule has a molecularweight greater than or equal to about 80,000 Da. In certain embodiments,a macromolecule has a molecular weight greater than or equal to about90,000 Da. In certain embodiments, a macromolecule has a molecularweight greater than or equal to about 100,000 Da.

In certain embodiments, the macromolecule is a naturally occurringmacromolecule. In certain embodiments, the macromolecule is a syntheticor semi-synthetic macromolecule. In certain embodiments, themacromolecule is present as part of a complex or conjugate with anothermolecule, e.g., a karyopherin.

In certain embodiments, the macromolecule is selected from the groupconsisting of RNA, mRNA, DNA, proteins, glycoproteins, carbohydrates,lipids, toxins, and any combination thereof.

In certain embodiments, the macromolecule is RNA.

In certain embodiments, the macromolecule is mRNA.

In certain embodiments, the macromolecule is DNA.

In certain embodiments, the macromolecule is a protein.

In certain embodiments, the macromolecule is a glycoprotein.

In certain embodiments, the macromolecule is a carbohydrate.

In certain embodiments, the macromolecule is a lipid.

In certain embodiments, the macromolecule is a toxin.

Having described the present invention in detail, the same will be moreclearly understood by reference to the following examples, which areincluded herewith for purposes of illustration only and are not intendedto be limiting of the invention.

EXAMPLES

Materials and Methods

DNA engineering:

The Nsp1³⁰⁻⁵⁹¹ gene from the Chait group was amplified by polymerasechain reaction (PCR) to prepare BamHI (B)-NheI (N)-Nsp1³⁰⁻⁵⁹¹-SpeI(S)-HindIII (H) and B-N-cNsp1 (Nsp1²⁸²⁻⁵⁸⁵)-S-H. Then, both DNAfragments were subcloned into the pET22b expression plasmid (C-terminal6×His tag (SEQ ID NO: 30)) using BamHI and HindIII restriction sites.The pET22b vector was chosen due to the C-terminal 6×His tag (SEQ ID NO:30) which allows isolation of full length proteins by metal affinitychromatography. B-N-1NLP-S-H, B-N-2NLP-S-H,B-P-EcoRI-intein-N-cNsp1-S-H, B-P domain (P)-N-cNsp1-S-P-H,B-P-N-1NLP-S-P-H and B-P-N-2NLP-S-P-H were also prepared. Gene sequencesof B-P domain (P)-N-1NLP_(1/2)-S-P-H and B-P-N-2NLP_(1/2)-S-P-H werepurchased (GenScript, USA) and subcloned into the pET22b expressionplasmid using BamHI and HindIII restriction sites. To prepare P-1NLP-Pand P-2NLP-P, N-1NLP_(1/2)-S and N-2NLP_(1/2)-S were subcloned intopET22b-B-P-N-1NLP_(1/2)-S—P-H and pET22b-B-P-N-2NLP_(1/2)-S-P-H plasmidsusing the SpeI restriction enzyme site. P-cNsp1-P was prepared bysubcloning N-cNsp1-S into the pET22b-B-P-N-1NLP_(1/2)-S-P-H plasmidusing NheI and SpeI restriction sites. 1NLP and 2NLP were prepared bysubcloning N-1NLP-S and N-2NLP-S from pET22b-B-P-N-1NLP-S-P-H andpET22b-B-P-N-2NLP-S-P-H plasmids into pET22b-B-P-N-1NLP_(1/2)-S-P-H andpET22b-B-P-N-2NLP_(1/2)-S-P-H plasmids using NheI and SpeI restrictionenzyme sites. B-P-EcoRI-intein-N sequences were purchased (IntegratedDNA Technologies, USA) and subcloned into the pET22b-B-N-cNsp1-S-Hplasmid using BamHI and NheI restriction enzyme sites. Sequences of allplasmids were confirmed by gene sequencing (Genewiz, USA). Preparedprotein sequences are as follows.

Nsp1³⁰⁻⁵⁹¹ (SEQ ID NO: 10) MDIGINSDPSTGAGAFGTGQSTFGFNNSAPNNTNNANSSITPAFGSNNTGNTAFGNSNPTSNVFGSNNSTTNTFGSNSAGTSLFGSSSAQQTKSNGTAGG NTFGSSSLFNNSTNSNTTKPAFGGLNFGGGNNTTPSSTGNANTSNNLFGATANANKPAFSFGATTNDDKKTEPDKPAFSFNSSVGNKTDAQAPTTGFSFGSQLGGNKTVNEAAKPSLSFGSGSAGANPAGASQPEPTTNEPAKPALSFGTATSDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSKPAFTFGKLAAALEHHHHHH cNsp1 (SEQ ID NO: 11) MDIGINSDPGSGSGASDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGA KPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSKPAFTFGTSGSGKLAAALEHHHH HH P-Intein-cNsp1(SEQ ID NO: 12)  MDIGINSDPAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASAISGDSLISLASTGKRVSIKDLLDEKDFEIWAINEQTMKLESAKVS RVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDELSLKEHIALPRKLESSSLQLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFVANDIIVHNASDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSKPAFTFGTSGSGKLAAALEHHHHHH 1NLP (SEQ ID NO: 13) MDIGINSDPGSGASPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPD EKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAF SFGAKPDEKKDDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSGSGKLAAALEHHHHHH  2NLP (SEQ ID NO: 14) MDIGINSDPGSGASPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSKTSGSGKLAAALEHHHHHH  P-cNsp1-P (SEQ ID NO: 15) MDIGINSDPAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEK KDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSKPAFTFGTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGKLAAALEHHHHHH  P-1NLP-P (SEQ ID NO: 16) MDIGINSDPAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDA SGASPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKP AFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVME SDASGGKLAAALEHHHHHH P-2NLP-P (SEQ ID NO: 17) MDIGINSDPAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKP AFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVME SDASGGKLAAALEHHHHHH 

Protein Expression and Purification:

All prepared genes were transformed into E. coli OverExpress C41(DE3)cells (Lucigen, USA). For expression, a freshly-grown bacterial colonywas inoculated in 10 mL LB medium with 100 μg/mL ampicillin at 37° C.overnight. 10 mL of overnight culture of all samples was inoculated into1 L terrific broth (TB) media at 37° C. with 100 μg/mL ampicillin untilOD₆₀₀˜1. Expression was then induced overnight at room temperature withthe addition of 1 mM IPTG. The cells were harvested, lysed in 50 mM Tris(pH 8), 300 mM NaCl and 8 M urea, and frozen at −80° C. Thawed cellswere sonicated, and 15 w/v % ammonium sulfate was added. Cell lysateswere clarified by centrifugation (14,000 g for 30 min at 4° C.), and theproteins of interest were purified by Ni-NTA affinity chromatographyunder denaturing conditions with 250 mM imidazole used for proteinelution. Purified samples were dialyzed against deionized (DI) water,and 20 mM Tris (pH 8) and 6 M urea were added. The proteins were furtherpurified by ion exchange chromatography using a HiTrap Q HP column (GEHealthcare, Sweden) in an ÄKTA pure FPLC. Samples containing the desiredproduct were dialyzed against DI water and lyophilized. P-intein-cNsp1was prepared using the same expression condition as the other proteins,but harvested cells were resuspended in 50 mM NaH₂PO₄, 300 mM NaCl, and10 mM imidazole and stored at −80° C. Thawed cells were lysed by thesonication, and cell lysates clarified by centrifugation were storedovernight at 4° C. at pH 7 for intein self-cleavage. Sun Z et al.,Protein Expr Purif 43: 26 (2005). After adjusting the pH to 8, sampleswith 6×His tags (SEQ ID NO: 30) were purified by Ni-NTA under nativeconditions and by FPLC as described above. P-C₃₀-P protein was expressedand purified as reported previously (Olsen B D et al., Macromolecules43: 9094 (2010)), with additional FPLC purification under denaturingconditions. Lyophilized samples were weighed to calculate yields ofsamples from 1 L culture (FIG. 2B and FIG. 3B). The purity of sampleswas determined to be greater than 95% by SDS-PAGE analysis (FIG. 2A andFIG. 3A).

For transport studies, pQE80-14×His tag-TEV-IBB-MBP-EGFP, pQE80-14×Histag-TEV-MBP-mCherry and pQE30-scImp β-6×His tag from the Görlich groupwere transformed into SG13009 (pRep4) cells, and expression followed apreviously described protocol. Frey S et al., EMBO J 28: 2554 (2009).After cutting off the His-tag with TEV protease (Eton Bioscience, USA)for IBB-MBP-EGFP and MBP-mCherry, additional purification was performedfor all three proteins by size exclusion chromatography in buffercontaining 50 mM Tris (pH 7.5) and 200 mM NaCl. The concentration ofrecombinant fluorescent proteins was determined using their opticalabsorbance.

FITC-Dextran:

Fluorescein isothiocyanate (FITC)-dextran with molar masses of 4, 10,40, 70 and 150 kg/mol (catalog #46994, FD10S, FD40, 46945 and 46946),were purchased from Sigma-Aldrich (USA). Approximate Stokes' radii ofthe FITC-dextran polymers were obtained from the supplier as follows:1.4 nm (4 kg/mol), 2.3 nm (10 kg/mol), 4.5 nm (40 kg/mol), 6.0 nm (70kg/mol) and 8.5 nm (150 kg/mol).

Hydrogel Preparation:

Lyophilized samples were dissolved at a concentration of 200 mg/mL in 50mM Tris/HCl (pH 7.5) and 200 mM NaCl except where otherwise noted,mixed, and allowed to gel at either room temperature or 4° C. It isknown that natural nucleoporins can form homogeneous hydrogels at suchhigh protein concentration, but at lower nucleoporin concentrations, thehydrogels lose their selective permeability function. For this reason,nucleoporin hydrogels, Nsp1, are typically prepared at 150-200 mg/mLconcentration throughout the literature. Since the invention concernsdeveloping artificially engineered protein hydrogels that mimic thefunction of natural nucleoporin hydrogels, the nucleoporin hydrogelswere benchmarked to these previous studies and used the concentrationwhere it showed the selective transport property. Having confirmed thatthe designed hydrogels at 20 w/v % can show selectivity (FIG. 4 and FIG.5), the ability of the best performing gel, P-2NLP-P, to mimic thisproperty at a lower gel concentration, 10 w/v % (FIG. 5), was checked.During the hydrogel inversion test, food coloring was added in thebuffer for better visualization of the test.

Rheology:

Oscillatory shear rheology was performed on an Anton Paar MCR-301 inDirect Strain Oscillation mode with TruGap™ control. A Peltier heatingsystem and environmental enclosure were employed to control sampletemperature. Samples were loaded into a 25 mm cone-and-plate geometrywith an angle of 1° and sealed with a mineral oil barrier to preventdehydration.

Raman Spectroscopy:

A custom-built NIR confocal Raman inverted microscopy system was usedfor Raman measurements. 785 nm light from a continuous-wave Ti:Sapphirelaser (3900S, Spectra-Physics) pumped by a frequency-doubled Nd:YAGlaser (Millennia 5sJ, Spectra-Physics) was used for the excitation. Awater immersion objective lens with 1.2 NA (UPLSAPO60XWIR 60×/1.20,Olympus) was used both to focus the laser onto the sample, which is ontop of quartz coverslip (043210-KJ, Alfa Aesar), and to collect thebackscattered light. The Rayleigh light in the collected signal wasremoved by dichroic mirrors (LPD01-785RU-25×36×1.1, Semrock). Ramanlight was delivered to an imaging spectrograph (Holospec f/1.8i, KaiserOptical Systems) and detected by a TE-cooled, back-illuminated, deepdepleted CCD (PIXIS: 100BR_eXcelon, Princeton Instruments). The laserpower at the sample plane was ca. 60 mW, and the signal was integratedfor 5 seconds. Nine spectra were collected from each sample andaveraged. Spectrum processing (cosmic ray removal, backgroundsubtraction and normalization) was performed by MATLAB (Mathworks)scripts.

Capillary Transport Measurements:

1.5 inch borosilicate capillaries with 0.9 mm inner diameters (Vitrocom8290) were loaded by piercing pre-made hydrogels. 5 μM solutions ofIBB-MBP-EGFP, MBP-mCherry, and/or importin β were injected into thecapillary and sealed by a 1:1:1 mixture of vaseline, lanolin, andparaffin. Time lapses of cargo transport into the hydrogels were takenat 1 minute intervals for 1-3 hours on a Nikon Ti Eclipse invertedmicroscope using a Nikon CFI Plan UW 2× and Hamamatsu C11440-22CUcamera. All fluorescence intensity profiles were obtained by averagingthe fluorescence intensity within 100 μm slice width through the centerof the gel and across the gel/buffer interface. The profiles werenormalized by the bath concentration in the capillaries at the firsttime point. The interface between gel and buffer is assigned by a 20%change in the intensity compared to the bath fluorescence intensity asthe zero point of the distance scale in each fluorescence intensityprofile.

Circular Dichroism Spectroscopy:

CD spectra were obtained on an Aviv Model 202 Circular Dichroismspectrometer. CD spectra were recorded in a quartz cell of 0.1 cm pathlength at 25° C. between 190 and 250 nm, using a scan rate of 12 nm/minwith a wavelength step of 1 nm. cNsp1 and the NLPs were dissolved in 50mM Tris buffer with 200 mM NaCl at pH 7.5. CD band intensities, afterthe buffer signal subtractions, were converted into mean residueellipticity (MRE).

Example 1: Preparation and Expression of Engineered Proteins

To reduce protein loss during washing step in Ni-NTA chromatographydenaturing purification, the washing buffer did not contain imidazoleand was prepared at pH 8, causing increased impurity during elution.After the elution, eluted proteins were run on denaturing gels (FIG. 6).While Nsp1 (60 kg/mol), one of nuclear pore proteins, was expressed atlow levels and difficult to identify on the gel, engineered proteins(P-cNsp1-P and P-NLPs-P) were highly expressed. For better purity, anionexchange chromatography was performed, with final purified productsshown in FIG. 2A. Note that all proteins ran slower in SDS-PAGE (FIGS.2A, 7A, and 6) than their calculated and measured molar masses (Table1), similar to previous reports for P-C₃₀-P protein. Olsen B D et al.,Macromolecules 43: 9094 (2010).

TABLE 1 Protein molar masses measured by MALDI cNsp1 1NLP 2NLP P-cNsp1-PP-1NLP-P P-2NLP-P Measured (g/mol) 35,623 36,756 36,223 46,305 46,33145,780 Calculated (g/mol) 35,460 36,461 36,329 45,971 45,885 45,437

TABLE 2 DNA plasmids Protein name Expressed protein Vector Nsp1³⁰⁻⁵⁹¹Nsp1-His₆ pET-22b cNsp1(=Nsp1²⁷⁴⁻⁵⁹¹) cNsp1-His₆ pET-22b cNsp1P-Intein-cNsp1-His₆ pET-22b 1NLP 1NLP-His₆ pET-22b 2NLP 2NLP-His₆pET-22b P-cNsp1-P P-cNsp1-P-His₆ pET-22b P-1NLP-P P-1NLP-P-His₆ pET-22bP-2NLP-P P-2NLP-P-His₆ pET-22b P-C₃₀-P His₆-P-C₃₀-P pQE-9   IBB-MBP-EGFPHis₁₄-TEV-IBB-MBP-mEGFP pQE-80   MBP-mCherry His₁₄-TEV-MBP-mCherrypQE-80   scImpβ scImpβ-His₆ pQE-30  

Engineered proteins with P domain blocks—P-cNsp1-P, P-1NLP-P andP-2NLP-P—were easily synthesized in much higher yield than recombinantnucleoporin Nsp1. After protein expression and chromatographicpurification, the yield of high purity protein was more than 20- to70-fold higher than the recombinant Nsp1 protein (FIG. 2 and FIG. 6).

NLPs without the coiled-coil domain were also isolated at 10 timesgreater yield than their parent sequence, cNsp1, after the sameprocedure (FIG. 3). Interestingly, when the cNsp1 was fused to the Pdomain endblocks (P-cNsp1-P), the construct was expressed at a similaryield as the NLP constructs.

Based on this observation, a single P domain together with an inteinself-cleavage domain (Mathys S et al., Gene 231: 1 (1999)) was subclonedinto the N-terminal cNsp1 gene (P-intein-cNsp1). After the self-cleavageof P-intein domains, cNsp1 was obtained at a similar yield as the NLPs(FIG. 3). Significantly improved biosynthetic yields of theseartificially engineered proteins enable detailed characterization oftheir material properties and engineering to control their performance.

Example 2: Preparation and Characterization of Hydrogels

Engineered proteins with P domain endblocks rapidly formed hydrogels,while NLP midblocks alone failed inversion tests, indicating thatstructure beyond the FG repeat is necessary to give elastic mechanicalproperties. Consistent with a previous study on recombinant cNsp1, theNLPs without associating coiled-coil domains did not pass hydrogelinversion tests, while the designed proteins with the P domains formedhydrogels within a few minutes, mainly limited by the time required forthe lyophilized sample to swell in a buffer. The engineered proteinswere found to gel in number of buffer conditions commonly used for therecombinant Nsp1, demonstrating that P domain endblocks successfullyreplace the role of the N-terminal sequences of Nsp1 as a gelcrosslinker.

More particularly, while cNsp1 is known not to form hydrogels, P-cNsp1-Pformed gels in all commonly used buffer conditions for Nsp1 at a proteinconcentration of 200 mg/mL. Buffers tested included (i) water; (ii) 0.1%TFA (in water), followed by neutralization with ¼ volume of the buffer(400 mM Tris-base, 100 mM Tris/HCl pH 7.5, 1 M NaCl); (iii) 0.1% TFA (inwater) and neutralized with ⅕ volume of 200 mM Tris-HCl (pH 8.5); (iv)0.2% TFA (in water) and neutralized with ⅕ volume of 200 mM Tris-HCl (pH9); and (v) 100 mM phosphate buffer.

Example 3: Significance of Midblock FG Repeats

To characterize the effect of midblock interactions on hydrogelmechanics, frequency sweep, linear oscillatory shear rheology of 20 w/v% hydrogels was performed in the absence or presence of 10% 1,6hexanediol. Measurements were performed at 25° C. with a strainamplitude of 1%, within the linear viscoelastic range. Representativeresults are shown in FIG. 8.

Rheology showed that the midblock sequence has a significant impact onthe low frequency viscoelastic properties of the triblock protein gelswithout affecting the high frequency elastic plateau modulus. Althoughthe midblocks cNsp1, 1NLP, and 2NLP are insufficient to cause gelationwithout the P domain, all three proteins with P domains formed gels witha comparable modulus (on the order of 10 kPa) with the crossover betweenG′ and G″ occurring below 0.1 rad/s (FIG. 7A-C). In all threeartificially engineered hydrogels, the addition of 10% hexanediol to a20 w/v % gel led to a decrease in the gel relaxation time, increasingthe crossover frequency of the gel by approximately a factor of 5, whilethe stiffness of hydrogels (the plateau modulus G′) changed very little(FIG. 7A-C).

Aliphatic alcohols, such as 1,6 hexanediol, are known to weaken FGassociations, leading to a loss of selective permeability in vivo and invitro. Comparison to a control hydrogel of similar molar mass butwithout FG repeats in the midblock (P-C₃₀-P) showed no effect on thecrossover frequency and the high frequency plateau modulus after theaddition of hexanediol (FIG. 7D and FIG. 9), indicating that theendblock P domains are unaffected by hexanediol. Therefore, the changesin crossover frequency in nucleoporin-mimetic gels, characteristic ofchanges in network relaxation rate, originate from differences in thestate of the midblock domain.

It has been shown that interchain β-sheets in some nucleoporin FG repeathydrogels contribute to crosslinking and enhance the FG hydrogelstability, and removing these crosslinks enhances permeability andreduces selectivity. Labokha A A et al., EMBO J 32: 204 (2013). It isexpected that the choice of crosslinking group in the hydrogel mayaffect biomolecular transport and mechanical properties, as crosslinkingcontrols the mesh size of the gel and the relaxation dynamics of thejunction points. These properties can influence the transport ofmacromolecules interacting with the network chains.

Prominent changes in Raman bands upon the addition of hexanediolconfirmed that these changes in gel mechanics are caused by disruptionof FG repeats involved in molecular interactions within the midblocks,indicating that naturally observed FG interactions have beensuccessfully adapted into the biosynthetic hydrogels. Upon the additionof hexanediol, a significant decrease was observed in the Raman band at486 cm⁻¹ corresponding to a Phe vibrational mode for cNsp1 and bothconsensus repeat NLP midblocks. Other Phe Raman bands (band assignmentsin FIG. 10) are similar for all midblock polymers in the presence andabsence of the hexanediol (FIG. 7E-G), indicating that natural cNsp1 andsynthetic NLPs have a similar physicochemical environment for the Pheresidues.

Other common changes upon the addition of hexanediol were observed inRaman bands at 685 and 710 cm⁻¹. These bands do not appear inlyophilized cNsp1 or NLP (FIG. 11) or in individual amino acids includedin NLPs in water solution from a previous study. Zhu G et al.,Spectrochim Acta A Mol Biomol Spectrosc 78: 1187 (2011). This suggeststhat the bands are a result of the association between midblocks inwater. Molecular interactions between Phe and CH₃ and Pro and Lys havebeen suggested in cNsp1 based upon Nuclear Overhauser ExchangeSpectroscopy NMR spectra by Ader et al., Proc Natl Acad Sci USA 107:6281 (2010). The addition of 10% hexanediol suppressed Raman bandsresponsible for Phe (486 cm⁻¹), Pro (856 and 1097 cm⁻¹), Lys (1442cm⁻¹), and CH₃ (1452 cm⁻¹) in cNsp1 (FIG. 7E), consistent with the NMRresult,^([24]) and therefore the observed Raman bands at 685 and 710cm⁻¹ may also relate to those residues.

The similar shifts in the crossover frequency in all designed hydrogels(FIG. 7A-C) and large intensity differences of the 486, 685 and 710 cm⁻¹(FIG. 7E-G) by the addition of hexanediol suggest that hydrophobicinteractions, including Phe-mediated associations, between the midblocksexist, similar to the natural Nsp1 hydrogel. These interactions caninfluence the gel relaxation without contributing significantly to theplateau modulus G′ (FIG. 7A-C).

Example 4: Transport Selectivity of Engineered Protein Hydrogels

Engineered protein hydrogels containing cNsp1, 1NLP, and 2NLP midblocksselectively enhanced transport of specific biomolecules into thehydrogels, mimicking the property of natural Nsp1 gels. A fluorescenceassay originally established to test recombinant nucleoporin hydrogelswas performed in a capillary geometry (FIG. 4A) to test whethercargo-NTR complexes can permeate through the engineered biosyntheticgels with enhanced transport accumulation, while other molecules andcargo without the NTR are significantly retarded.

For the assay, importin β (95 kg/mol) was chosen as a NTR due to itswell-known binding to cargo with an importin β binding (IBB) domain andto the FG repeat on nucleoporin hydrogels. To reduce the passage ofcargo without the NTR and easily quantify the transport of selectedcargo, recombinant IBB—maltose binding protein (MBP)—enhanced greenfluorescent protein (EGFP) protein fusions were prepared as a modelcargo protein (75 kg/mol; Stokes' radii of MBP and GFP are reported as2.85 nm and 2.42 nm, respectively.). Based on the widely applied dextrandiffusion method, it was expected that cargo diffusion into the gelwould be significantly reduced since the pore size of the gels issmaller than non-interacting 40 kg/mol dextran probe (4.5 nm of Stokes'radius; FIG. 12). When importin β and the cargo were physically mixedand added to the capillary prepared with the engineered hydrogels,selective partitioning into the hydrogel occurred over time, while aslab diffusion profile was detected in the absence of importin β (FIG.4B-E and FIG. 13). The enhanced transport into the gel occurred due to acombination of diffusion and convection caused by gel swelling withbuffer and cargo complexes (FIG. 14).

Because the length scale of the measurement was much larger than themolecular size and gel mesh size, the gel can be considered as auniform, semi-infinite slab. The gels can also be treated asmacroscopically homogeneous since they are optically clear (absence ofinhomogeneity that would scatter on the length scale of visible light)and did not phase separate upon centrifugation. Under these conditions,the permeability coefficient is the product of the diffusivity andsolubility coefficients. The discontinuous concentration profile at theinterface during the capillary experiments suggested that thecargo-importin β complexes are more soluble in the gel phase than thecargo alone because of the physical association between importin β andFG repeats. This increase in solubility enhances the permeability of thecargo complexes.

Example 5: Electrostatic Effects on Transport Properties

The two simplified NLP midblocks, both consensus sequences of cNsp1 butdiffering by a single amino acid in the repeating peptide, showedquantitative differences in transport properties. When accumulated greenfluorescence intensities were calculated compared to the intensitieswithout importin β (FIG. 4F and FIG. 13), the P-2NLP-P gel showed almosttwice the intensity of the P-1NLP-P gel. P-cNsp1-P, P-1NLP-P, andP-2NLP-P hydrogels absorbed 3.9±0.4 (mean±SD, n=3), 2.3±0.1 (n=3), and3.8±0.3 (n=6) times more cargo-importin β complexes than inert moleculesin an hour, respectively. Both NLPs have equal numbers of FG repeats,the same P domain crosslinkers for gelation, similar secondary structureas determined by circular dichroism (FIG. 15 and Table 3), and similarpassive diffusion profiles for inert molecules over time (dotted blackcurves in FIG. 4D-E). Therefore, this quantitative change inpermeability is believed to originate from the change in the consensusrepeat sequence.

TABLE 3 CD analysis of cNsp1 and NLPs^(a) Protein Helix 1 Helix 2 Strand1 Strand 2 Turns Unordered Total NRMSD cNsp1 0.00 0.02 0.18 0.11 0.140.54 0.99 0.125 1NLP 0.00 0.02 0.19 0.12 0.18 0.47 0.98 0.141 2NLP 0.010.02 0.18 0.12 0.18 0.48 0.99 0.128 ^(a)By the circular dichroismsecondary structure (CDSSTR) method (Kang JW et al., Biomed Opt Express2: 2484 (2011)), it was found that approximately 50% structures of allproteins were disordered and others were β-strand and β-turns.

Since the single amino acid change Asp in 1NLP to Ser in 2NLP occurredin the middle of the peptide between FG repeats that are known to bindimportin β, the change from an anionic to neutral residue (change from aformal charge of −16 to 0 for the entire midblock of 16 repeats)suggests that electrostatic effects may affect molecular transport. Itis interesting to note that the hydrogel made from P-cNsp1-P, where themidblock has a formal charge of +6, shows higher cargo-carrieraccumulation on the gel interface than the P-2NLP-P hydrogel (max.fluorescence intensity: 7.6±0.7 and 5.0±0.9 for P-cNsp1-P and P-2NLP-P,respectively). However, the depth-integrated accumulation in an hour isthe same for both materials (FIG. 4F), indicating that the P-2NLP-P gelhas better permeability to the cargo-carrier than P-cNsp1-P gel.

Changes in the charge of the protein based on a single substitution perrepeat unit between 1NLP and 2NLP affect biomolecular transport throughthe designed hydrogels, despite the use of high ionic strength buffersthat screen charge as under physiological conditions. Recent studies ofrelated biological hydrogels such as mucus and cartilage have similarlyobserved that electrostatic effects influence molecular transport at thephysiologically relevant ionic strength conditions. Selective bindingcan be added to synthetic systems by conjugating FG peptide onto polymergels. The results presented here on NLP hydrogels indicate that not onlyFG sequences but also residues far from the FG repeat can play animportant role in the performance of nuclear pore-mimetic synthetichydrogels.

Example 6: Further Characterization of P-2NLP-P

After identifying P-2NLP-P as the top performing construct, additionalexperiments were performed to explore its performance. The addition of10% hexanediol to disrupt FG interactions eliminated selectiveaccumulation of cargo complexes in P-2NLP-P gels (FIG. 4G), showing thatFG repeat interactions are involved in enhanced selective transport inthe biosynthetic hydrogels. This indicates that the engineered hydrogelshave a filtering mechanism similar to the natural nucleoporin system.

Using blends of model proteins with and without the IBB domainestablished the ability of the biosynthetic NLP gels to activelytransport cargo proteins compared to the inert proteins. A model cargoprotein incapable of binding importin β, MBP-mCherry (67 kg/mol), hassmaller size than IBB-MBP-EGFP (75 kg/mol) but showed retarded transportthrough the biosynthetic NLP hydrogels even in the presence of importinβ (FIG. 4H-I, 3 hours assay in 10 w/v % and 20 w/v % of P-2NLP-P gelresults in FIG. 5).

FIG. 4H-I shows selective permeability test performed on P-1NLP-P andP-2NLP-P biosynthetic hydrogels (20 w/v %) with the addition of 5 μMMBP-mCherry, a model inert molecule, into 5 μM IBB-MBP-EGFP/importin βcargo complex mixtures. Over an hour, the cargo-carrier complexesaccumulated 3.0 and 5.3 times more than MBP-mCherry (without the IBBdomain) in P-1NLP-P and P-2NLP-P hydrogels, respectively.

When hydrated in buffer at 20% and 10%, P-2NLP-P formed optically-cleargels that did not phase separate upon centrifugation, suggesting theyformed macroscopically homogeneous networks under these conditions. FIG.5 shows that over three hours, the cargo-carrier complexes accumulatedin the gel 10.2 and 5.2 times more than MBP-mCherry (no IBB domain),indicating that the 10 w/v % gel still showed enhanced transport of theselected biomolecules and the total accumulation depended on the numberof FG sequences in the hydrogels (i.e., there are twice as many FGsequences in the 20 w/v % gel compared to the 10 w/v % gel).

The results illustrate that the designed hydrogels can mimic both theselectivity and enhanced transport of natural nucleoporin hydrogels.

Example 7: NTR-Mediated Selective Uptake of a Target Molecule

This example illustrates a generalizable method for capturing selectedmolecules into a hydrogel of the invention. As shown in FIG. 18, apeptide tag which can associate with a target molecule of interest canbe genetically fused to a nuclear transport receptor (NTR). In asolution, the peptide tag fused to NTR will recognize, i.e., associatewith, its target molecule and thereby spontaneously form NTR-peptidetag-target molecule complexes. Due to the interaction between NTR andthe hydrogel, the NTR-peptide tag-target molecule complex is captured byand carried into the hydrogel.

As a model system, incomplete green fluorescent protein (GFP) was usedas a target molecule and GFP tag as a binding peptide tag. It is knownthat complete GFP can be constructed by mixing incomplete GFP and theGFP tag, with green fluorescence as evidence of the assembly. CabantousS et al., Nat Methods 3: 845-854 (2006); Kent K P et al., J Am Chem Soc130: 9664-9665 (2008). Another benefit using GFP as a model is thatgreen fluorescence can be used to visualize selective transport of theNTR-GFP tag-incomplete GFP (i.e., NTR-GFP) complex into the hydrogelunder a fluorescence microscope.

GFP tag was genetically fused to the C-terminus of the NTR nucleartransport factor 2 (NTF2). Since NTF2 is homodimer, each NTF2 has twoGFP tags. For protein purification, 6×Histidine tag (SEQ ID NO: 30) wasalso fused between NTF2 and GFP tag (NTF2-His tag-GFP tag). Afterprotein synthesis, more than 50 mg of chimeric NTF2-GFP tag wasobtained.

Capillary transport assay validated the method and confirmed that thehydrogel system can capture selected molecules into the hydrogel. WhenNTF2 with GFP tag was mixed with incomplete GFP, the solution turnedfrom no color to light green after overnight incubation. When thesolution was added to one end of a capillary where 20 wt % hydrogelfilled the other end (FIG. 19), the green fluorescence was accumulatedonto the hydrogel over time (FIGS. 19A-19C). In the same experimentalconditions, fluorescein-labeled dextran (40 kg/mol, hydrodynamic radius:4.5 nm) did not get into the hydrogel (FIGS. 19D-19F).

One or two incomplete GFP can associate with NTF2-GFP tag homodimerwhich has two GFP tags. The molar mass of GFP is approximately 30 kg/mol(2.42 nm hydrodynamic radius). The molar mass of NTF2-GFP tag homodimeris 36 kg/mol. Since NTF2-GFP complex (66 kg/mol with one GFP, or 96kg/mol with two GFP) is greater than the 40 kg/mol dextran, the resultsclearly show that the synthetic system can mimic the natural selectivefiltering nuclear pore function which is carrying target molecule intothe gel although the size of the NTF2-GFP complex is greater thanuncomplexed inert reference molecules.

The method just described opens new avenues for numerous applicationsfor a number of applications, including for example drug delivery, foodtoxicology, and defense. Researchers have developed peptide library forvarious target molecules using phage display, and solid phase peptidesynthesis techniques. By simply fusing those peptides to NTR carriers,the carriers will capture the targets and bring them into the hydrogels.More and diverse peptides will be available for specific targetmolecules with time; thereby, the method will be further generalized. Asa specific example, Staphylococcal enterotoxin B (SEB) toxoid can becaptured into the hydrogel using anti-SEB tag fused to NTF2 or importinβ; the hydrogel can then be removed or destroyed for environmentaldecontamination.

INCORPORATION BY REFERENCE

All patents and published patent applications mentioned in thedescription above are incorporated by reference herein in theirentirety.

EQUIVALENTS

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

We claim:
 1. A polypeptide comprising a plurality of contiguousinstances of the sequence of PAFSFGAKPDEKKDSDTSK (SEQ ID NO: 1), whereinthe polypeptide forms a hydrogel.
 2. The polypeptide of claim 1, furthercomprising a first leucine zipper domain endblock, wherein the firstleucine zipper domain endblock flanks the N-terminal end at theplurality of contiguous instances of the sequence of SEQ ID NO:1 orflanks the C-terminal end of the plurality of contiguous instances ofthe sequence of SEQ ID NO:1.
 3. The polypeptide of claim 2, wherein thefirst leucine zipper domain endblock consists of a pentamericcoiled-coil domain (P domain).
 4. The polypeptide of claim 3, whereinthe P domain consists of the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).
 5. Thepolypeptide of claim 1, further comprising a first leucine zipper domainendblock flanking the N-terminal end of the plurality of contiguousinstances of the sequence of SEQ ID NO:1; and a second leucine zipperdomain endblock flanking the C-terminal end of the plurality ofcontiguous instances of the sequence of SEQ ID NO:1.
 6. The polypeptideof claim 5, wherein at least one of the first or second leucine zipperdomain endblock consists of a P domain.
 7. The polypeptide of claim 6,wherein the P domain consists of the peptide ofAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).
 8. Thepolypeptide of claim 1, comprising the sequence:APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:4).
 9. A polypeptidecomprising a plurality of contiguous instances of the sequence ofPAFSFGAKPDEKKDDDTSK (SEQ ID NO: 2), wherein the polypeptide forms ahydrogel.
 10. The polypeptide of claim 9, further comprising a firstleucine zipper domain endblock, wherein the first leucine zipper domainendblock flanks the N-terminal end of the plurality of contiguousinstances of the sequence of SEQ ID NO:2 or flanks the C-terminal end ofthe plurality of contiguous instances of the sequence of SEQ ID NO:2.11. The polypeptide of claim 10, wherein the first leucine zipper domainendblock consists of a pentameric coiled-coil domain (P domain).
 12. Thepolypeptide of claim 11, wherein the P domain consists of the peptide ofAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).
 13. Thepolypeptide of claim 9, further comprising a first leucine zipper domainendblock flanking the N-terminal end of the plurality of contiguousinstances of the sequence of SEQ ID NO:2; and a second leucine zipperdomain endblock flanking the C-terminal end of the plurality ofcontiguous instances of the sequence of SEQ ID NO:2.
 14. The polypeptideof claim 13, wherein at least one of the first or second leucine zipperdomain endblock consists of a P domain.
 15. The polypeptide of claim 14,wherein the P domain consists of the peptide represented byAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3).
 16. Thepolypeptide of claim 9, comprising the sequence:APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:5).
 17. A nucleicacid molecule encoding the polypeptide of claim
 1. 18. An expressionvector comprising the nucleic acid molecule of claim
 17. 19. A cell,comprising the expression vector of claim
 18. 20. A hydrogel, comprisingthe polypeptide of claim
 1. 21. A filtering device, comprising thehydrogel of claim 20; and a housing or support for the hydrogel.
 22. Adrug delivery device, comprising a drug; and the hydrogel of claim 20.23. A method of separating or selectively filtering macromolecules,comprising contacting a source of macromolecules with a hydrogel ofclaim 20.